We are using systematic experimental and computational approaches to map the molecular networks induced by DNA damage. Since DNA damage response pathways are conserved across eukaryotes, our studies are focused on the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe-impacting our basic knowledge of the DNA damage response while keeping within species for which systems data are easily obtained. In our past five years of funding (NIH grant R01-ES14811), significant progress was made in mapping the architecture of transcriptional networks responding to DNA damage and in perturbing these networks to reveal their key state transitions. In the present proposal, we turn our attention to the global networks of DNA-damage-induced kinases and other signaling proteins. Specific Aim 1 will use Epistatic MiniArray Profiling (E-MAP) to develop dynamic genetic interaction maps of signaling across a panel of ten diverse DNA damaging agents. Genetic interaction screens will be conducted among a core set of ~500 genes including all kinases, phosphatases, and transcription factors (TFs) in budding and fission yeast. Specific Aim 2 will use expression profiling to identify functional interactions between kinases and TFs in response to DNA damage. Kinase/TF pairs that regulate similar damage-responsive genes will be investigated for functional association by seeking specific phosphorylation sites on the TF and exploring the functional consequences of TF phosphorylation on DNA repair. Specific Aim 3 will seek to computationally integrate the interaction data to identify conserved network modules and to experimentally categorize these modules by their specific functions related to DNA repair. Network maps will be visualized and processed using Cytoscape and deposited in the CellCircuits database. Developing a comprehensive map of DNA-damage-induced genetic interactions will be critical for understanding the genetic polymorphisms that confer susceptibility to DNA damage. Such networks are also a predictive tool for synthetic lethality and epistasis in human cancer. PUBLIC HEALTH RELEVANCE: We propose to develop a comprehensive genetic and physical interaction map of signal transduction in response to DNA damage. This map is a major biomedical resource which will be used to identify and target chemotherapeutic agents and their modulators.